Our main research efforts are directed toward a better understanding of the structural and functional properties of RNAs, the enzymes that modify these molecules, and the involvement of GTP in protein biosynthesis. Our experimental approach involves the use of analogs of nucleic acid components. These are generally incorporated into the RNAs, the RNAs isolated and their properties studied. The uracil analog 5-fluorouracil (FU) has been used as a probe in a number of laboratories, including our own. In this study we propose to use FU-containing tRNAs as substrates to probe the active site of ribothymidine synthetase from E. coli, and FU-containing 5S RNAs to explore the potential of 19F-N.M.R. spectroscopy in studying the structure of FU-containing RNAs and perhaps ribonucleoproteins in solution. Transfer RNAs isolated from E. coli grown in the presence of 2-thiouracil (SU) serve nicely as substrates for pseudouridine synthetase II in vitro (1,2). These substrates combined with the tritium-release assay (3), afford an excellent method for following the purification of pseudouridine synthetase II. Once isolated in a more highly purified form, the properties of this enzyme will be examined in some detail. Photoaffinity analogs of nucleic acid components have recently been employed in a number of biological systems and promise to be powerful biochemical probes. We intend to use two of these, 5-azido-CTP and 8-azido-ATP, to examine the binding site(s) of nucleotidyl transferase. The mode of action of GTP in protein biosynthesis will be studied through the use of the photo-sensitive 8-azido-GTP analogs. Finally, we intend to investigate whether or not a variety of these photoaffinity analogs can be incorporated into tRNA using nucleotidyl transferase and polynucleotides using polynucleotide phosphorylase. Macromolecules containing highly reactive light-sensitive azido compounds, would be extremely useful in studying a variety of complexes--including supramolecular ones.